J. Agric. Food Chem. 2001, 49, 4961−4964
4961
Improved Method for the Synthesis of trans-Feruloyl-β-sitostanol Anthony M. Condo, Jr.,† David C. Baker,*,† Robert A. Moreau,‡ and Kevin B. Hicks‡ Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600, and Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038
Phytosterols and phytostanols are known to lower low-density lipoprotein-cholesterol (LDL-C) levels in humans by up to 15%, and at least two products, Benecol and Take Control, are now on the market as naturally derived fatty acid esters of phytostanols (stanol esters) and phytosterols (sterol esters), respectively. A synthetic process was developed to synthesize gram quantities of transferuloyl-β-sitostanol from ferulic acid and β-sitostanol, with high purity and yields of ∼60%. The process involves (a) condensation of trans-4-O-acetylferulic acid with the appropriate phytostanol or phytostanol mixture in the presence of N,N-dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine, (b) separation of the trans-4-O-acetylferuloyl products by preparative liquid chromatography, (c) selective deacetylation of the feruloyl acetate, and (d) chromatographic purification of the feruloylated phytostanols. The process was successfully applied to synthesize stanol trans-feruloyl esters from “Vegetable Stanols”, a mixture of ∼70:30 β-sitostanol and β-campestanol, in comparable purity and yield. Keywords: β-Sitostanol; trans-feruloyl-β-sitostanol; phytostanols; stanol esters INTRODUCTION
Two of the most popular cholesterol-lowering “functional” foods currently on the market are Benecol and Take Control, with the former containing synthetic fatty acid esters of phytostanols (stanol esters) derived from tall oil and the latter containing synthetic fatty acid esters of phytosterols (sterol esters) derived from soybean oil (1). Phytosterols and phytostanols are known to lower low-density lipoprotein-cholesterol (LDL-C) levels in humans by up to 15%, and the FDA has recently permitted a rare health claim for their use in low-fat diets (2). Phytosterols and phytostanols are thought to lower LDL-C by inhibiting the absorption of cholesterol from the intestine (3). Free phytosterols and phytostanols are poorly soluble in most foods, and this has led to problems with formulations and inconsistencies in efficacy. To solve this problem, they are usually esterified with fatty acids to make them soluble in margarines and other high-fat food products (4). In the intestine, fatty acid esters are hydrolyzed by digestive enzymes, producing the physiologically active free sterol or stanol (4). We at the USDA laboratories have recently discovered and patented a natural phytosterol-containing oil from corn fiber (corn fiber oil) that contains high levels of natural phytosterol and phytostanol fatty acid esters and a unique class of stanol esters, the stanol ferulate esters (also called ferulate phytostanol esters) (5, 6). Although many clinical studies have been performed on fatty acid esters of stanols and sterols, no studies have examined the efficacy of stanol ferulate esters. Because these compounds contain a ferulic acid moiety, which is a powerful antioxidant, it is proposed that in addition to reducing LDL-C levels, they might * Corresponding author [telephone (865) 974-1066; fax (865) 974-1536; e-mail
[email protected]]. † University of Tennessee. ‡ U.S. Department of Agriculture.
also reduce the oxidation of LDL-C, further lowering the risk of coronary heart disease. To test this hypothesis, quantities of pure stanol ferulate esters are required. A method that uses 4-O-acetylferuloyl chloride as the acylating agent has been published for the synthesis of these types of esters (7, 8). The present study was undertaken to develop an improved method for the synthesis of β-sitostanol ferulate (5a). Because “soy stanols” are a more readily available commercial product, containing ∼70% β-sitostanol (3a) and 30% β-campestanol (3b), the ferulate ester synthesis was also evaluated with this mixed substrate (3a + 3b). The published procedures (7, 8) for the chemical synthesis of trans-feruloyl-β-sitostanol is limited by two steps: (1) the required preparation of the highly reactive trans-4-O-acetylferuloyl chloride, which is difficult to purify and handle, and (2) the deprotection step that uses sodium borohydride to remove the acetyl protective group on the trans-feruloylated product. Our goal in the present work was to develop a more workable synthesis of the compounds. MATERIALS AND METHODS General Procedures. All reactions were conducted under a nitrogen atmosphere unless otherwise indicated. Anhydrous solvents were dried prior to distillation as follows: dichloromethane, chloroform, and pyridine were refluxed over calcium hydride; THF was refluxed over sodium benzophenone ketyl; and methanol was refluxed with magnesium metal shavings. trans-Ferulic acid and acetic anhydride were purchased from Aldrich Chemical Co. and used without further purification. β-Sitostanol (3a) was purchased from Research Plus, Inc., and used without further purification. To maximize the yield of the coupled products 4a and 4b, the starting material was dried for 18 h to remove residual ethanol (78 °C,
